I Molecular basis for Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2. Normally, the eukaryotic type II topoisomerases (Top2 and Top2) regulate DNA topology by employing a dsDNA cleavage and religation cycle involving transient formation of Top2-DNA cleavage-complexes (Top2cc). Top2 catalytic intermediates are characterized by the topoisomerase covalently linked to the DNA 5-terminus by an active site tyrosine residue. However, aberrant DNA structure or targeted chemotherapeutic disruption of the Top2 reaction can generate Top2cc, protein-DNA crosslinks that block transcription and/or collapse DNA replication forks. Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. To understand Tdp2 functions, we determined X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation- interface. We have further identified a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. II Structure of Sirtuin-linked Macrodomains Cells use the post-translational modification ADP-ribosylation to control a host of biological activities. In some pathogenic bacteria, an operon-encoded mono-ADP-ribosylation cycle mediates response to host-induced oxidative stress. In this system, reversible mono ADP-ribosylation of a lipoylated target protein represses oxidative stress response. An NAD+-dependent sirtuin catalyzes the single ADP-ribose (ADPr) addition, while a linked macrodomain-containing protein removes the ADPr. We have been studying the structure and function of macrodomain protein, and determined the crystal structure of the sitruin-linked macrodomain protein from Staphylococcus aureus, SauMacro (also known as SAV0325) to 1.75- resolution. The monomeric SauMacro bears a previously unidentified Zn2+-binding site that putatively aids in substrate recognition and catalysis. An amino-terminal three-helix bundle motif unique to this class of macrodomain proteins provides a structural scaffold for the Zn2+ site. Structural features of the enzyme further indicate a cleft proximal to the Zn2+ binding site appears well suited for ADPr binding, while a deep hydrophobic channel in the protein core is suitable for binding the lipoate of the lipoylated protein target. III Structure and function of the Ape2 nuclease The Xenopus laevis APE2 nuclease dictates 3-5 nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. We have determined that APE2 resection activity is modulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DNA damage response proteins Topoisomerase 3 and NEIL3 DNA glycosylase, as well as transcription and RNA regulatory proteins such as TTF2, TFIIS and RPB9. Biochemical and NMR results establish the nucleic acid binding activity of the Zf-GRF domain. Further, APE2 X-ray structures and small angle X-ray scattering (SAXS) analyses show that the Zf-GRF fold is typified by a crescent shaped ssDNA binding claw, that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3-5 exonuclease processing, as well as prevent efficient APE2-dependant RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module dictating the key ssDNA strand break resection function of APE2, and further reveal topological similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.